1. Field of the Invention
The present invention is in the field of adjuvants and immunostimulating agents. More particularly, the invention pertains to novel triterpene saponin-lipophile conjugates.
2. Related Art
Saponins are glycosidic compounds that are produced as secondary metabolites. They are widely distributed among higher plants and in some marine invertebrates of the phylum Echinodermata (ApSimon et al., Stud. Org. Chem. 17:273-286 (1984)). Because of their antimicrobial activity, plant saponins are effective chemical defenses against microorganisms, particularly fungi (Price et al., CRC Crit. Rev. Food Sci. Nutr. 26:27-135 (1987)). Saponins are responsible for the toxic properties of many marine invertebrates (ApSimon et al., Stud. Org. Chem. 17:273-286 (1984)). The chemical structure of saponins imparts a wide range of pharmacological and biological activities, including some potent and efficacious immunological activity. In addition, members of this family of compounds have foaming properties (an identifying characteristic), surfactant properties (which are responsible for their hemolytic activity), cholesterol-binding, fungitoxic, molluscicidal, contraceptive, growth-retarding, expectorant, antiinflammatory, analgesic, antiviral, cardiovascular, enzyme-inhibitory, and antitumor activities (Hostettmann, K., et al., Methods Plant Biochem. 7:435-471(1991); Lacaille-Dubois, M. A. & Wagner, H., Phytomedicine 2:363-386 (1996); Price, K. R., et al., CRC Crit. Rev. Food Sci. Nutr. 26:27-135 (1987)).
Structurally, saponins consist of any aglycone (sapogenin) attached to one or more sugar chains. In some cases saponins may be acylated with organic acids such as acetic, malonic, angelic and others (Massiot, G. & Lavaud, C., Stud. Nat. Prod. Chem. 15:187-224(1995)) as part of their structure. These complex structures have molecular weights ranging from 600 to more than 2,000 daltons. The asymmetric distribution of their hydrophobic (aglycone) and hydrophillic (sugar) moieties confers an amphipathic character to these compounds which is largely responsible for their detergent-like properties. Consequently, saponins can interact with the cholesterol component of animal cell membranes to form pores that may lead to membrane destruction and cell death, such as the hemolysis of blood cells. ##STR1##
Saponins can be classified according to their aglycone composition as shown above:
1. Triterpene glycosides
2. Steroid glycosides
3. Steroid alkaloid glycosides
The steroid alkaloid glycosides, or glycoalkaloids, share many physical and biological properties with steroid glycosides, but alkaloid glycosides are usually considered separately because their steroidal structure contains nitrogen. Frequently, the aglycones have methyl substituents that may be oxidized to hydroxymethyl, aldehyde or carboxyl groups; these moieties may play a role in some of the saponin's biological activities. From extensive studies of saponins, it is apparent that the triterpene saponins are not only the most predominant in nature, but also those with the most interesting biological and pharmacological properties.
Saponins have one or more linear or branched sugar chains attached to the aglycone via a glycosidic ether or ester link. In some saponins, the presence of acylated sugars has also been detected. According to the number of sugar chains attached to the aglycone, the saponins can be monodesmosidic saponins (with a single sugar chain), or bidesmosidic saponins (with two sugar chains). In the monodesmosidic saponins, the sugar chain is typically attached by a glycosidic ether linkage at the C-3 of the aglycone. In addition to the C-3 linked sugar chain, bidesmosidic saponins have a second sugar chain bound at C-28 (triterpene saponins) or at C-26 (steroid saponins) by an ester linkage. Because of the typical liability of esters, bidesmosidic saponins are readily converted into their monodesmosidic forms by mild hydrolysis (Hostettmann, K., et al., Methods Plant Biochem. 7:435-471 (1991)) (FIG. 2). Apparently, monodesmosidic saponins are significantly more biologically active in plants than their bidesmosidic forms. For instance, in Hedera helix the enzymatic transformation of the bidesmosidic hederasaponin C to its monodesmosidic form (.alpha.-hederin) results in a product with a high antibiotic activity (Wagner, H. & Horhammer, L., Pharmacognosy and Phytochemistry, Springer-Verlag, Berlin (1971)). In general, monodesmosidic saponins also tend to be more hemolytic than bidesmosidic saponins. This property appears to correlate well with their antifungal activity. Presumably, by interacting with the fungi's membrane-bound sterols, saponins alter the permeability of cell membranes leading to the organism's death (Price, K. R., et al., CRC Crit. Rev. Food Sci. Nutr. 26:27-135 (1987)). Consequently, the host range of plant pathogenic fungi appears to be functionally determined by their capacity to enzymatically detoxify the host organism's saponins (Bowyer, P., et al., Science 267:371-374 (1995)). However, the acylated quillaja saponins appear to be exceptional since their monodesmosidic forms are significantly less effective hemolytic agents than their acylated and non-acylated bidesmosidic forms (Pillion, D. J., et al., J. Pharm. Sci., 84:1276-1279 (1996)). Bidesmosidic saponins most likely function as useful forms for storage and/or transport of these compounds until such time as the biologically active monodesmosidic forms are required for the plant's defense (Hostettmann, K., et al, Methods Plant Biochem. 7:435-471 (1991); Osbourn, A. E., et al., Adv. Exp. Med. Biol., 404:547-555 (1996)). In contrast, in animals, bidesmosidic saponins may have potent biological and pharmacological activities that are completely unrelated to any aspects of plant physiology.
Saponin adjuvants from the bark of the Quillaja saponaria Molina tree (Quillajasaponins) are chemically and immunologically well-characterized products (Dalsgaard, K. Arch. Gesamte Virusforsch. 44:243 (1974); Dalsgaard, K., Acta Vet. Scand. 19 (Suppl. 69):1(1978); Higuchi, R. et al., Phytochemistry 26:229 (1987); ibid. 26:2357 (1987); ibid. 27:1168 (1988); Kensil, C. et al., J. Immunol. 146:431 (1991); Kensil et al., U.S. Pat. No. 5,057,540 (1991); Kensil et al., Vaccines 92:35 (1992); Bomford, R. et al., Vaccine 10:572 (1992); and Kensil, C. et al., U.S. Pat. No. 5,273,965 (1993)).
These saponin adjuvants are a family of closely related O-acylated triterpene glycoside structures. They have an aglycone triterpene (quillaic acid), with branched sugar chains attached to positions 3 and 23, and an aldehyde group in position 23. A unique characteristic of the Quillajasaponins is the presence of acyloil acyl moieties linked at the C-3 hydroxy group of a fucopyranose bound by an ester bond to position 28 of quillaic acid. These acyl moieties have been identified as 3,5-dihydroxy-6-methyloctanoic acid, 3,5-dihydroxy-6-methyloctanoic acid 5-O-.alpha.-L-rhamnopyranosyl-(1.fwdarw.2)-.alpha.-L-arabinofuranoside, and 5-O-.alpha.-L-arabinofuranoside.
Higuchi, R. et al. (Phytochemistry 26:229 (1987); ibid. 27:1168 (1988), and Kensil, C. et al. (U.S. Pat. No. 5,057,540, ibid., Vaccine 92:35 (1992), and U.S. Pat. No. 5,273,965 (1993)) have demonstrated that the 3-O-glycosidic linkage between the fucosyl residue and the acyloil acyl residue can be cleaved by mild alkaline hydrolysis to yield desacylsaponins. These desacylsaponins from Quillajasaponins are more hydrophilic than the original saponins. Apparently, deacylation of Quillajasaponins results in a significant loss of adjuvant activity, as measured by antibody production and CTI response (Kensil et al., U.S. Pat. No. 5,057,540 at column 22, lines 35 to 49; Kensil et al., Vaccines 92:35 (1992); and Kensil et al., U.S. Pat. No. 5,273,965, column 7, line 62).
Quillajasaponins are found as a mixture of about twenty structurally closely related triterpenoid glycosides with minimal differences between them (Higuchi, R. et al., Phytochemistry 26:229 (1987); ibid., 26:2357 (1987); ibid., 27:1169 (1988); Kensil et al., U.S. Pat. No. 5,057,540 (1991); Kensil et al., Vaccines 92:35 (1992)), making their separation difficult. Their triterpenoid group carries the aldehyde group responsible for inducing T-cell immunity, whereas their carbohydrate moieties seem to enhance humoral immunity (perhaps by interacting with lymphocyte receptors) in a fashion similar to certain polysaccharides (Bohn J. and J. BeMiller, Carbohydrate Polymers 28:3 (1995). In effect, PCT published application WO 90/03184 describes that saponins with their triterpenoid aldehyde reduced to alcohol are still able to induce an antibody response. Another component of quillajasaponins, the acyloil-acyl groups, likewise appear to play a role in adjuvanticity. There are also reasons to suspect that their acyloil acyl moiety, formed by a normoterpene carboxylic acid, is in part responsible for some of the toxic properties observed with several of the purified Quillajasaponins (Kensil, C. et al., J. Immunol. 146:431 (1991)). Thus, it would be of commercial interest to develop modified Quillajasaponins which are easier to purify, potentially less toxic, chemically more stable, and with equal or better adjuvant properties than the original saponins.
The immune system may exhibit both specific and nonspecific immunity (Klein, J., et al., Immunology (2nd), Blackwell Science Inc., Boston (1997)). Generally, B and T lymphocytes, which display specific receptors on their cell surface for a given antigen, produce specific immunity. The immune system may respond to different antigens in two ways: 1) humoral-mediated immunity, which includes B cell stimulation and production of antibodies or immunoglobulins [other cells are also involved in the generation of an antibody response, e.g. antigen-presenting cells (APCs; including macrophages), and helper T cells (Th1 and Th2)], and 2) cell-mediated immunity (CMI), which generally involves T cells including cytotoxic T lymphocytes (CTLs), although other cells are also involved in the generation of a CTL response (e.g., Th1 and/or Th2 cells and APCs).
Nonspecific immunity encompasses various cells and mechanisms such as phagocytosis (the engulfing of foreign particles or antigens) by macrophages or granulocytes, and natural killer (NK) cell activity, among others. Nonspecific immunity relies on mechanisms less evolutionarily advanced (e.g., phagocytosis, which is an important host defense mechanism) and does not display the acquired nature of specificity and memory, hallmarks of a specific immune response. Nonspecific immunity is more innate to vertebrate systems. In addition, cells involved in nonspecific immunity interact in important ways with B and T cells to produce an immune response. The key differences between specific and nonspecific immunity are based upon B and T cell specificity. These cells predominantly acquire their responsiveness after activation with a specific antigen and have mechanisms to display memory in the event of future exposure to that specific antigen. As a result, vaccination (involving specificity and memory) is an effective protocol to protect against harmful pathogens.
A critical component of inactivated vaccines, including subunit vaccines, is an adjuvant. Adjuvants are nonimmunogenic compounds, that when administered with an antigen (either mixed with, or given prior to the administration of the antigen) enhances or modifies the immune response to that particular antigen. Thus, the humoral and/or cell-mediated immune responses are more effective when an antigen is administered with an adjuvant. Furthermore, the adjuvant may alter the quality of the immune response by affecting the subclasses (isotypes) of imnmunoglobulins produced (IgG1, IgG2, IgG3, and IgG4 for human IgGs; IgG1, IgG2a, IgG2b, and IgG3 for mouse IgGs), as well as their affinities. A response regulated by Th1 cells in mice will induce IgG I, IgG2a, IgG2b and to a lesser extent IgG3, and also will favor a CMI response to an antigen. If the IgG response to an antigen is regulated by Th2 type cells it will predominantly enhance the production of IgG1 and IgA.
Adjuvants that have been used to enhance an immune response include aluminum compounds (all generally referred to as "alum"), oil-in-water emulsions (often containing other compounds), complete Freund's adjuvant (CFA, an oil-in-water emulsion containing dried, heat-killed Mycobacterium tuberculosis organisms), and pertussis adjuvant (a saline suspension of killed Bordatella pertussis organisms). These adjuvants generally are thought to have their mechanism of action by causing a depot of antigen and permitting a slow release of the antigen to the immune system, and by producing nonspecific inflammation thought to be responsible for their observed activity (Cox, J. C., et al., Vaccine 15:248-256 (1997)). Some saponins have been shown to have different types of immune stimulating activities, including adjuvant activity. These activities have been reviewed previously (Shibata, S., New Nat. Prod. Plant Pharmacol. Biol. Ther. Act., Proc. Int. Congr. 1st, 177-198 (1977); Price, K. R., et al. CRC Crit. Rev. Food Sci. Nutr. 26:27-135 (1987); Schopke, Th., & Hiller, K., Pharmazie 45:313-342 (1990); Lacaille-Dubois, M. A., et al., Phytomedicine 2:363-386 (1996)).
PCT published application WO 93/05789 describes conjugates in which poorly immunogenic proteins are covalently attached to purified, acylated Quillaja saponin fraction via the carboxyl group of 3-O-glucuronic acid. Addition of free quillajasaponins to these conjugates induced a higher immune response suggesting (I) that the covalently attached quillajasaponin serves as an association site for additional saponin molecules and (ii) that the adjuvant effect depends on the number of saponins associated with the protein antigen.
PCT published application WO 90/03184 describes an immunostimulating complex (ISCOM) comprising at least one lipid and at least one saponin, and that may optionally include adjuvants in addition to the saponin. These matrices are taught to be useful as immunomodulating agents and vaccines. The lipid and saponin are in a physical association, rather than covalently attached to one another. Quil A (a Quillaja saponin extract) is the preferred saponin. The reference additionally teaches that it is beneficial to add adjuvants (in addition to Quil A) to the ISCOM matrix. The reference teaches that an adjuvant lacking suitable hydrophobic properties may be modified to comprise a hydrophobic domain for incorporation into the ISCOM matrix.
Bomford, R. et al., Vaccine 10:572-577 (1992) teaches that lipids can be mixed with a variety of saponins to form ISCOM's. The reference teaches that Quillaja saponins, Gypsophila saponins and Saponaria saponins were the only saponins tested that were adjuvant active.
There remains a need for adjuvants that have enhanced adjuvanticity and lower toxicity.